ABSTRACT/PROJECT SUMMARY From a human health perspective, genetic heterogeneity due to accumulation of mutations in normal tissues increases cancer risk, and is likely an important factor in many degenerative diseases, such as adult macular degeneration, congestive heart failure, inflammatory & autoimmune disorders, sarcopenia & muscle weakness, and skeletal & joint degeneration. Somatic mutations can occur due to DNA damage or replication errors. However, it has been difficult to detect somatic mutations in individual patients because it has not been possible to reliably compare sequences of the genomes of single cells. Human colon crypts can be used as a clonal representation of the colon stem cell at the base of each crypt. Using SNP microarray, we have shown that large-scale human somatic cell-to-cell differences (deletions, gene conversions, duplications) exist in colon crypts and increase with age. We have optimized methods for whole genome sequencing without whole genome amplification to >30X depth using a single colon crypt of 1000 to 2000 cells to study mutations accumulated in both nuclear and mitochondrial genomes. An increase in single base mutations in both the nuclear and mitochondrial genomes with age was observed in the whole genome sequencing analysis. Based on power calculations using these preliminary findings, we propose to expand the study to include a total of 21 individuals distributed among three age groups to statistically validate our findings and examine potential mechanisms for the age-related DNA damage. In Aim 1, whole sequencing libraries will be constructed from five single colon crypts and the bulk tissue for >30X coverage of the genome from each individual human subject over a range of ages from children to elderly. In Aim 2, the whole genome sequencing data will be examined for point mutations and small indels, and for crypt to germline and crypt-to-crypt changes in the same individual. In Aim 3, large deletions will be identified to discern the mechanism of both the DNA breakage and the repair. In Aim 4, DNA sequence motifs at sites of genetic change will be examined to identify preferred damage sites in the nuclear as well as the mitochondrial genome. The potential biochemical basis and the molecular repair pathway of these age- related DNA aberrations will also be explored. These efforts are important for understanding what DNA sequences are at highest risk for the types of DNA damage or error-prone DNA repair that result in accumulation of genetic change. This study will lay the foundation for future work to study how environment and diet may affect the progression of somatic genetic changes over the human lifespan as well as how polymorphisms may predispose some individuals to more rapid degenerative diseases or cancer and thus permit consideration of preventive measures. Also, as diagnostic analysis relies upon progressively fewer somatic cells, this knowledge of somatic cell genetic heterogeneity will become increasingly important for precision medicine.